Our research plan is focused on generating a comprehensive understanding of G-protein-coupled receptors (GPCRs) and their signaling partners on epithelial stem cell proliferation and differentiation. For the first year we are establishing the following projects: 1- Comprehensive identification of GPCRs involved in epithelial cell proliferation and differentiation. I have identified the GPCRs expressed in epithelial cells. This information will be used to identify particular GPCRs regulating skin stem cell proliferation and differentiation by a loss of function focused-screen using RNA interference (RNAi) and CRISPER-CAS9 sgRNA. GPCRs identified to have an effect on epithelial stem cell proliferation or differentiation will be blocked or over-activated by genetic and/or pharmacological intervention and RNA sequencing will be performed. This data will help elucidate the signaling pathways and transcriptional networks affected by specific GPCRs and G proteins in epithelial cells. The biological relevance of these additional downstream pathways will then be explored. 2- Define the role of G alpha proteins on stem cell biology. By using animal models and 3D in vitro organotypic cultures of skin we are exploring how G alpha proteins regulate epithelial stem cell biology. In particular, we are exploring the possibility that activation of G alpha i, which opposes G alpha s signaling by reducing cAMP production, might participate in the regulation of skin stem cells. In order to study the effect of activating G alpha i GPCR signaling in the skin, we are taking advantage of human muscarinic designer receptors (DREADDs). We are expressing these receptors in the skin and inducing their activation to study the effect of G alpha i activation. In addition, we are taking advantage of other DREADDs to study additional G alpha proteins. 3- Development of a mechanistic signaling framework for the regulation of epithelial stem cells by GPCRs and G proteins. This aim focuses on the characterization of the cytoplasmic and nuclear events downstream of GPCRs and G proteins involved in epithelial stem cell regulation. In order to analyze how GPCR signaling affects long term epigenetic events that regulate epithelial stem cell differentiation, we are focusing on how G alpha s-PKA signaling control the activity of EZH2, a master epigenetic regulator. In addition, we are focusing on studying other proteins downstream from GPCRs that affect long term survival and proliferation of epithelial stem cells.